Cloning of putative odorant-degrading enzyme and integumental esterase cDNAs from the wild silkmoth, Antheraea polyphemus
Cloning of putative odorant-degrading enzyme and integumental esterase cDNAs from the wild silkmoth, Antheraea polyphemus
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DOI：
10.1016/s0965-1748(02)00136-4
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发表时间：
2002-12-01
影响因子：
3.8
通讯作者：
Leal, WS
中科院分区：
文献类型：
--
作者：
Ishida, Y;Leal, WS
气味降解酶被认为参与昆虫信息素的快速失活。这些蛋白质在昆虫触角的感觉淋巴中特异性表达，表达量如此之低，以至于迄今为止，分离和基于蛋白质的cDNA克隆是不可能的。根据昆虫羧酸酯酶和保幼激素酯酶的保守氨基酸序列设计简并引物，扩增出部分cDNA片段，并设计了RACE基因特异性引物。这种生物信息学方法使我们克隆了编码野生蚕蛾（Antheraea polyphemus）的推定气味降解酶（Apol-ODE）和推定外皮酯酶（Apol-IE）的cDNA。Apol-ODE的预测分子量为59，994 Da，pI为6.63，具有三个潜在的N-糖基化位点，以及一个推定的催化位点Ser，其特征在于序列Gly（195）-Glu-Ser-Ala-Gly-Ala。Apol-IE的计算分子量为61，694 Da，pI为7.49，有两个潜在的N-糖基化位点，以及一个序列为Gly（214）-Tyr-Ser-Ala-Gly的推定活性位点。RT-PCR检测到Apol-ODE基因在雄虫触角和触角分枝（感器组织）中的转录，而在雌虫触角和其它对照组织中均未检测到。Apol-IE在雌雄触角和腿中均有检出。(C)2002爱思唯尔科技有限公司。保留所有权利。
Odorant-degrading enzymes have been postulated to participate in the fast deactivation of insect pheromones. These proteins are expressed specifically in the sensillar lymph of insect antennae in such low amounts that, hitherto, isolation and protein-based cDNA cloning has not been possible. Using degenerate primers based on conserved amino acid sequences of insect carboxylesterases and juvenile hormone esterases, we were able to amplify partial cDNA fragments, which were then used for the design of gene-specific primers for RACE. This bioinformatics approach led us to the cloning of cDNAs, encoding a putative odorant-degrading enzyme (Apol-ODE) and a putative integumental esterase (Apol-IE) from the wild silkmoth, Antheraea polyphemus. Apol-ODE had a predicted molecular mass of 59,994 Da, pI of 6.63, three potential N-glycosylation sites, and a putative catalytic site Ser characterized by the sequence Gly(195)-Glu-Ser-Ala-Gly-Ala. Apol-IE gave calculated molecular mass of 61,694 Da, pI of 7.49, two potential N-glycosylation sites, and a putative active site with the sequence Gly(214)-Tyr-Ser-Ala-Gly. The transcript of Apol-ODE was detected by RT-PCR in male antennae and branches (sensillar tissues), but not in female antennae and other control tissues. Apol-IE was detected in male and female antennae as well as legs. (C) 2002 Elsevier Science Ltd. All rights reserved.